The sensory quality of a yeast fermented beverage such as beer depends largely on the particular brewing yeast strain which is used. During the beer brewing process, the yeast will in addition to ethanol produce a large variety of minor metabolites which affect the taste and flavour of the beer. Whereas the presence of certain of such minor metabolites may, at least up to certain levels, confer desirable sensory characteristics to the beer, several minor metabolites are undesired and may even, if present in higher amounts, result in spoilage of the beer.
Yeast fermented beverages including beer contain more than a hundred minor metabolites, of which some, at least when they are present above certain concentrations, will confer to the beverage an undesirable taste and flavour. A comprehensive review of beer flavouring compounds has been given by Meilgaard (1975) to which there is referred. In this connection, the most important metabolites include sulphur compounds such as dimethyl sulphide (DMS), hydrogen sulphide (H.sub.2 S), thiols and thioesters which at certain levels will result in undesirable sulphur-containing flavours.
DMS is a thioether of great importance for the aroma and flavour of beer. The content of DMS in conventional lager beers regularly exceeds the taste threshold level of about 30 .mu.g/L (Meilgaard, 1975) which explains the focus that has been on this compound. Above its threshold level but below about 100 .mu.g/L DMS contributes to the distinctive taste of some lager beers. When present at concentrations above 100 .mu.g/L DMS may, however, impart a generally undesired flavour described as "cooked sweet corn". DMS in beer may be derived from thermal degradation of S-methyl-methionine during kiln-drying and wort preparation and it has been suggested that this is the only pathway of significance for the final DMS content in beer (Dickenson and Anderson, 1981; Dickenson, 1983). However, substantial evidence suggests that enzymatic conversion of dimethyl sulphoxide (DMSO) to DMS by the brewing yeast is of great importance and, under some circumstances, even the major source of the final DMS level in beer (Leemans et al., 1993).
There is no doubt, that Saccharomyces strains do contain an enzymatic activity that can reduce DMSO to DMS in a NADPH-dependent manner (Zinder and Brock, 1978a; Anness et al., 1979; Anness, 1980). A multicomponent methionine sulphoxide (MetSO) reductase (EC 1.8.4.5) has been isolated from yeast (Black et al., 1960; Porque et al., 1970), and it has been suggested that this activity is identical to the DMSO reductase activity (Anness et al., 1979; Anness, 1980; Bamforth, 1980; Bamforth and Anness, 1979; 1981). The normal function of MetSO reductase seems to be reduction of oxidized methionines of cellular proteins which is in agreement with observations showing that the enzyme has a higher affinity for MetSO than for DMSO (Bamforth and Anness, 1979; 1981) and that MetSO inhibits DMSO reduction (Anness et al., 1979; Anness, 1980; Bamforth and Anness, 1981). The consequence hereof is that the degree of MetSO formation during kiln-drying of the malt will affect the degree of DMSO reduction.
The nitrogen content of the growth medium also seems to affect DMS formation by yeast. Thus, high amounts of highly assimilable nitrogen keep DMSO reductase activity at a low level, whereas enzyme activity is induced under nitrogen-limiting conditions (Gibson et al., 1985). The high nitrogen content of most worts would appear to keep DMSO reduction at the base level during fermentation. See Anness and Bamforth (1982) for a review on DMS formation in beer production. So-called DMSO reductases from some prokaryotes have been characterized, as well as the genes encoding them (Satoh and Kurihara, 1987; Bilous et al., 1988; Weiner et al., 1992; Yamamoto et al., 1995). While such enzymes, in some cases, have MetSO reducing capabilities, their main purpose in the bacterial cell is probably to act as the terminal step in DMSO respiration (Zinder and Brock, 1978b). Peptide methionine sulphoxide reductases (PMSRs) probably fulfil the role of repairing oxidised methionine species in proteins, thereby restoring their biological activity.
Sulphite is another sulphur-containing metabolite being formed during yeast fermentation. Sulphite is a versatile food additive used for preservation of foodstuffs. In beer sulphite has a dual activity in that it acts both as an antioxidant and as an agent that masks certain off-flavours. Due to its great importance, considerable work has been carried out to elucidate the physiology of sulphur metabolism in Saccharomyces species in relation to sulphite production.
Sulphite is produced by the yeast during beer fermentation and is present in the final beer. The compound is produced via the sulphur assimilation pathway. Inorganic sulphate is taken up through two sulphate permeases encoded by SUL1 and SUL2, respectively (Cherest et al., 1997). Intracellularly, sulphate is converted to adenylylsulphate (APS) by the action of ATP sulphurylase (EC 2.7.7.4) encoded by MET3. In a subsequent step, the MET14 encoded APS kinase (EC 2.7.1.25) catalyses the formation of phosphoadenylylsulphate (PAPS) which in turn is reduced by PAPS reductase (EC 1.8.99.4) (MET16 encoded) to sulphite.
Hydrogen sulphide results from reduction of sulphite and in this form inorganic sulphur is incorporated into organic compounds by fusion with O-acetyl homoserine leading to the formation of homocysteine. The latter compound is the precursor for biosynthesis of cysteine, methionine and S-adenosylmethionine (SAM). SAM represses transcriptionally all MET-genes (Cherest et al., 1985; Sangsoda et al., 1985; Langin et al., 1986; Thomas et al., 1989; Thomas et al., 1990; Korch et al., 1991; Mountain et al., 1991; Hansen et al., 1994).
Hydrogen sulphide is, as it is mentioned above, an intermediate in the sulphur assimilatory pathway of Saccharomyces spp. It is also the point of entry into the methionine biosynthetic pathway of carbon backbones derived from the threonine biosynthetic pathway. It is derived in four enzymatic steps from inorganic sulphate ions obtained from the growth medium of the yeast.
Besides being an intermediate in the yeast sulphur metabolism, hydrogen sulphide is an important flavour compound in beer and its distinct taste of "putrefied eggs" generally renders this compound undesired in beers, except at very low concentrations, where it may aid in disguising the taste of other flavour compounds such as acetyl esters.
Several workers have attempted to control the formation of hydrogen sulphide in beer production by modifying yeast strains by recombinant DNA technology. Thus, overexpression of the MET25 gene encoding the enzyme (EC 4.2.1.22) that catalyses the condensation of hydrogen sulphide and O-acetyl homoserine leads to a reduced hydrogen sulphide concentration in beer (Omura et al., 1995). When the gene NSH5 (STR4, CYS4) encoding the first of two steps (EC 4.2.1.22) in formation of cysteine from homocysteine is overexpressed, the hydrogen sulphide level is also reduced (Tezuka et al., 1992). This effect was suggested by these authors to be due to faster removal of homocysteine and thus of its precursor, hydrogen sulphide. These experiments suggest the existence of metabolic bottlenecks around the conversion of hydrogen sulphide into organic sulphur compounds. In the literature, there have been speculations that hydrogen sulphide may be a precursor for certain thiols and thioesters, e. g. methane- and ethanethiol (MeSH and EtSH) and methyl- and ethylthioacetate (MeSAc and EtSAc). Thus, in the above experiment with NSHS (Tezuka et al., 1992), the decrease in hydrogen sulphide production was followed by a decrease in methanethiol and ethanethiol production.
Another group of flavour compounds which, when present above certain threshold levels, result in undesirable flavours include higher alcohols and esters such as ethyl acetate, isoamyl acetate and ethyl hexanoate. Above their threshold levels such esters will confer a fruity (apple, banana) flavour to beer.
A variety of higher alcohols is found in beer. As used herein "higher alcohols" indicate other alcohols than ethanol. Such higher alcohols that are also referred to in the art as fusel alcohols are listed in Meilgaard (1975) and include isoamyl alcohol.
Some of the higher alcohols are produced by metabolism of the branched chain amino acids isoleucine, valine and leucine. .alpha.-Ketoacids (.alpha.-keto-.beta.-methylvalerate, .alpha.-ketoisovalerate and .alpha.-ketoisocaproate) are important intermediates for branched-chain amino acids as well as for higher alcohols. Isoamyl alcohol and its corresponding acetate ester are among the distinct beer flavour components.
Three enzymatic steps encoded by the genes LEU4 (Baichwal et al. 1983), LEU1 and LEU2 are involved in de novo synthesis of .alpha.-keto-isocaproate. The enzymes are .alpha.-isopropylmalate (.alpha.IPM) synthase (Leu4p, EC 4.1.3.12), isopropylmalate isomerase (Leulp, EC 4.2.1.33) and .beta.-isopropylmalate dehydrogenase (Leu2p, EC 1.1.1.85) (Reviewed by Kohlhaw, 1988). The pathway is regulated by a complex consisting of the regulatory protein Leu3p and isopropylmalate. The complex acts as an activator and regulates the level of mRNA being produced by binding to a regulatory region in front of the LEU4, LEU1 and LEU2 coding regions. Conversion of .alpha.-ketoisocaproate to leucine is catalyzed by the transaminases encoded by the genes BAT1 and BAT2. Deletion of both BAT1 and BAT2 results in auxotrophy for the branched-chain amino acids isoleucine, valine and leucine (Kispal et al. 1996).
In the yeast cell, the leucine level is regulated in several ways, of which feed-back inhibition of the isopropylmalate synthase by leucine is one. It is possible to inactivate the feed-back inhibition of Leu4p. Leucine feed-back inhibition resistant mutants with a dramatically lower sensitivity for leucine have been isolated. The mutants are resistant to the leucine analogue 5,5,5-trifluoro-DL-leucine (TFL). LEU4 was originally isolated as a mutant resistant to TFL (Baichwal et al. 1983). Such dominant feed-back resistant (LEU4.sup.fbr) mutations are known to produce high amounts of isoamyl alcohol and isoamyl acetate in brewers yeast (Lee et al. 1995).
Previous studies have shown that at least three isopropylmalate synthases are present in the yeast cell (Baichwal et al. 1983). Two synthases are produced by LEU4 (80% of the wild-type activity). The two forms produced by LEU4 include a long form (designated Ia) that is exported to the mitochondria and a short form designated Ib present in the cytoplasm. The gene coding for the remaining activity was originally designated LEU5, but later investigations have shown LEU5 to be a gene with PET gene similarities (petites: unable to grow on non-fermentable carbon sources) (Drain and Schimmel, 1986) and not directly involved in the leucine synthesis pathway. Inactivation of the LEU4 gene product does not in itself lead to leucine auxotrophy (Baichwal et al. 1983). The leu4 (leaky) phenotype might be due to other synthase activities. Three other loci (LEU6, LEU7 and LEU8) responsible for leucine biosynthesis have been described, where LEU7 and LEU8 appear to be candidates for a gene or genes that encode an .alpha.-IPM synthase activity (Drain and Schimmel, 1988). Linkage to known open reading frames has not yet been established.
During the Saccharomyces cerevisiae genome-sequencing project (http://genome-www.stanford.edu/Saccharomyces/), a putative LEU4 homologue has been identified. The open reading frame (ORF) is designated YOR108W and is located on chromosome XV. The ORF YOR108W is homologous to LEU4 with more than 80% nucleotide identity in a contiguous sequence of 1806 nucleotides of 1860 nucleotides found in the LEU4 ORF, whereas the upstream regulatory regions are clearly different. There appears to be a possibility for translation of a long and a short form similar to LEU4. Upstream of the YOR108W ORF two regulatory Gcn4p and Leu3p binding sequences seem to be present.
An .alpha.-ketoisocaproate decarboxylase activity encoded by the ORF YDL080C has been described for S. cerevisiae (Dickinson et al., 1997). This ORF is most likely the same as THI3 (Nishimura et al., 1992). The enzyme appears to catalyze the conversion of .alpha.-ketoisocaproate to isoamyl alcohol. In a ydl080 disruptant a minor activity (40% activity relative to that of the wild type) appears to be present.
Two alcohol acetyltransferase genes designated ATF1 (ORF YOR377W on chromosome XV) and ATF2 (ORF YGR177C on chromosome VII) have been identified in S. cerevisiae. Alcohol acetyl-transferases (AATases EC 2.3.1.84) catalyze the transfer of the acetyl group from acetyl-CoA to alcohols thereby producing acetate esters. AATasel encoded by the ATF1 gene has been purified (Malcorps & Dufour,1992 and Minetoki et al., 1993), while AATase2 is only known as an open reading frame, ATF2 (Nagasawa et al., 1995). AATasel and AATase2 have about 36% amino acid sequence identity. The ATF1 gene has been disrupted (Fujii et al. 1996b). This mutant produced about 20% isoamyl acetate and about 60% ethyl acetate as compared to the original strain (Fujii et al. 1996b).
The allotetraploid lager yeast, Saccharomyces carlsbergensis, has at least two different genomic sets. One genomic set is similar to that of S. cerevisiae whereas another genomic set is similar to that of S. monacensis (Pedersen, 1986). Allotetraploid lager yeast thus has two copies of a S. cerevisiae-like allele of ATF1 (designated herein as ATF1-CE), and two copies of a S. carlsbergensis specific allele (designated herein as ATF1-CA), presumably originating from S. monacensis. Fujii et al. (1996a) cloned the two genes ATF1-CE and ATF1-CA (previously designated ATF1 and Lg-ATF1, respectively) and determined the nucleotide sequence. The amino acid sequences encoded by the two genes are 76% identical. The ATF2-CE gene from the bottom fermenting yeast Saccharomyces pastorianus has been cloned and sequenced (Yoshimoto et al., 1996a, 1996b). It is not known whether two forms of this gene are found in the S. carlsbergensis lager yeast. Fermentation of wort with a brewers yeast transformed with a 2 .mu.-based plasmid containing the ATF1-CE gene resulted in increased levels of isoamyl acetate (7.6-fold) and ethyl acetate (3.9-fold) in the final beer compared to the control (Fujii et al., 1993).
Abnormal flavours may also be associated with various ketones of which diacetyl is the most important and with aldehydes such as acetaldehyde, the precursor for ethanol, and so-called staling aldehydes of which the most important is trans-2-nonenal which confer to beer a highly undesired "cardboard" flavour.
Currently, the brewing industry attempts to control the sensory quality of beer such as lager by several measures, including blending of batches having an undesired content of one or several metabolites with batches having a lower content hereof. However, when using conventional strains of brewer's yeast, it may not be possible to obtain a fully acceptable blend of beer by this approach. An alternative approach is to select, by using classical breeding and mutagenization techniques, yeast strains which, relative to the conventionally used strains, have a lower production of an undesired metabolite.
The most widely used yeast for brewing is Saccharomyces spp. However, not all Saccharomyces spp. are suitable for brewing purposes. Typical faults of non-brewing Saccharomyces strains include production of phenolic off-flavour, inability to utilize maltotriose and low fermentation rate at the temperature optimal for the desired aroma of the beer.
In the industry, the characteristics of the existing brewing yeasts may be improved by selecting strains which e.g. have a higher rate of fermentation, a decrease in beer maturation time, better flocculation characteristics or increased tolerance to alcohol.
There are, however, two major factors that currently limit progress in the breeding of brewing yeasts. First, it is often complicated to translate the desired change in yeast performance into biochemical and genetic terms. The second difficulty is that brewing yeasts generally have deficiencies in their sexual reproduction and as a consequence hereof it is difficult to carry out many of the breeding steps and procedures in genetic analysis that are trivial with the non-brewing yeasts used as genetic reference strains in academic studies.
Although the designation Saccharomyces carlsbergensis is widely accepted, this common lager yeast is also conventionally referred to as S. pastorianus or S. uvarum. It has a complicated genetic structure, being allotetraploid with one chromosome set similar to the S. cerevisiae genome while the other set is structurally similar to that of S. monacensis. Therefore, it is expected to find two copies of a S. cerevisiae-like allele and two copies of a S. monacensis-like allele of a certain gene in S. carlsbergensis. Hereinafter, the S. cerevisiae-like allele is specified by the gene name followed by -CE (e.g. MET2-CE). Similarly, the S. carlsbergensis-specific (S. monacensis-like) allele is designated by the gene name followed by -CA. It is generally assumed that this "mixed" genome may have an effect on the characteristics of the lager brewing yeast. Strain improvement by classical genetic methods is not straightforward, as most brewing yeasts exhibit poor sporulation and low spore viability.
It is therefore currently a significant problem in the brewing industry that it is impossible or difficult to provide a range of variants of the same beverage type such as lager beer which are specifically adapted to consumer preferences in individual marketplaces or to different seasons of the year by having specific and predetermined contents of compounds determining the sensory characteristics of the beverages.
Methods to make fermented beverages having a modified content of aroma or flavour compounds have been developed. One approach which has been used to modify beverages is to add isolated and/or concentrated aroma or flavour compounds derived from a yeast fermentation process and using such compounds as flavour additives to beverages. Thus, as an example, it is suggested in U.S. Pat. No. 3,713,838 to produce food products and beverages to which are added flavour compounds isolated from yeast dregs from a conventional brewing process. In WO 96/39480 is disclosed the use of kettle hop extracts to provide a fully hop flavoured beverage
Another approach is to provide yeast strains which have been modified to produce an increased level of particular flavour compounds. Thus, EP 574 941-Al discloses Saccharomyces cerevisiae strains transformed with a plasmid-borne gene coding for alcohol acetyltransferase (AATase). Such transformed strains having multiple copies of this AATase-encoding ATF1 gene showed an enhanced production of isoamyl acetate and ethyl acetate.
In WO 94/08019 is disclosed microbial strains which are transformed with a gene coding for .alpha.-acetolactate decarboxylase, an enzyme capable of converting the diacetyl precursor, .alpha.-acetolactate, directly to acetoin, thus avoiding the formation of diacetyl.
JP 62-92577 and 30-07579 disclose mutants of Saccharomyces cerevisiae that have enhanced production of amyl alcohol and isobutyl alcohol, whereas JP 50-49465 discloses yeast mutant strains producing high amounts of .beta.-phenethylene alcohol and esters thereof.
There have been very few attempts to provide brewing yeast strains including S. carlsbergensis having a reduced production of flavour compounds. Thus, it has been attempted to control the production of diacetyl by blocking the acetolactate synthase activity in Saccharomyces cerevisiae by mutation or in Saccharomyces carlsbergensis by in vitro deletion and replacement by recombination of ILV2 genes with the mutant alleles. However, completely blocking this biosynthetic step in Saccharomyces carlsbergensis has not yielded brewing strains with satisfactory characteristics, presumably because the parental strains herefor do not take up isoleucine and valine as effectively as Saccharomyces cerevisiae (Kielland-Brandt et al. 1995).
Thus, the prior art is not aware of industrially suitable strains of brewer's yeast in which pathways for production of undesired metabolites have been substantially completely interrupted or of strains of the lager brewing yeast Saccharomyces carlsbergensis that has been modified to produce in the brewing process none or less of a flavour compound and/or a compound that stabilizes a flavour compound.
It has now been found that it is possible to provide modified Saccharomyces brewer's yeast strains including modified strains of Saccharomyces carlsbergensis in which one or several biosynthetic pathways leading to the formation of flavour compounds or compounds stabilizing such compounds have been substantially completely blocked and that such modified strains have retained the beverage fermentation capacity and efficiency of the parent strains.
This has provided industrially attractive means for producing yeast fermented beverages including lager beers which substantially do not contain one or more flavour compounds or flavour stabilizing compounds that is/are normally produced by the lager yeast strain. In turn, this achievement has made it possible to produce, in an industrial production scale, a fermented base beverage in which one or more flavour-related metabolite(s) normally present in a particular beverage type is/are absent or present at very low levels. Such a base beverage can in turn be "flavour customized" by blending it with batches of beverage having a normal or enhanced content of the respective flavour compounds or by adding isolated flavour compounds to provide a composite beer having a desired, predetermined flavour compound profile.